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Method for catalyzing chenodeoxycholic acids to compound ursodesoxycholic acids through efficient whole-cells

A technology of ursodeoxycholic acid and whole cells, applied in the field of bioengineering, can solve problems such as difficult realization and complex process

Active Publication Date: 2016-03-02
苏州天绿生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the substrate 7-KLCA used in the above-mentioned different enzymatic synthesis reactions of UDCA still relies on the chemical oxidation of chenodeoxycholic acid; free enzymes are used in the reactions, because the whole reaction requires the participation of various enzymes and coenzymes, Makes the whole process more complicated and difficult to realize in industrial production

Method used

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  • Method for catalyzing chenodeoxycholic acids to compound ursodesoxycholic acids through efficient whole-cells
  • Method for catalyzing chenodeoxycholic acids to compound ursodesoxycholic acids through efficient whole-cells
  • Method for catalyzing chenodeoxycholic acids to compound ursodesoxycholic acids through efficient whole-cells

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Effect test

Embodiment 1

[0068] Escherichia coli BL21(DE3) containing recombinant DNA pET21a(+)-LDH-7αHSDH was inoculated into a 250mL Erlenmeyer flask containing 50mL of LB medium (100μg / mL ampicillin), cultivated overnight at 37°C and 300 rpm, and then 10 % of the inoculum was transferred into a 2L shake flask containing 400 mL of LB medium (100 μg / mL ampicillin), and continued to culture at 37° C. and 300 rpm for 2 hours until the OD600 reached 1.0. Add IPTG at a final concentration of 0.2 mM, induce expression at 25°C for 4 hours, and collect cells by centrifugation. The cells were suspended in 20 mL of 100 mM phosphate buffer (pH 8.0), sonicated, and centrifuged to obtain a recombinant mixed crude enzyme solution. The enzyme activities of 7α-HSDH and LDH were 747.0 units / mL and 1083 units / mL, respectively.

Embodiment 2

[0070] Escherichia coli BL21(DE3) containing recombinant DNA pET21a(+)-LKADH-7βHSDH was subjected to shake flask fermentation using the same method as in Example 1. The induction time was 4 hours. Cells were harvested by centrifugation. The cells were suspended in 20 mL of 100 mM phosphate buffer (pH 8.0), sonicated and centrifuged to obtain a recombinant mixed crude enzyme solution. The enzyme activities of 7β-HSDH and LKADH were 131.8 units / mL and 32.0 units / mL, respectively.

Embodiment 3

[0072] Inoculate a single colony on the Escherichia coli BL21(DE3) plate containing recombinant DNA pET21a(+)-LDH-7αHSDH into two 1L shake flasks filled with 250mL LB medium (100μg / mL ampicillin), at 37°C and 300rpm Incubate with shaking for 15 hours. Combine the seed liquids in the two shake flasks into 500mL and inoculate them into 5L of initial medium containing: 30g / L glycerol, 25g / L potassium dihydrogen phosphate, 10g / L ammonium sulfate , 10g / L magnesium sulfate heptahydrate, 0.4g / L iron sulfate heptahydrate. Recombinant Escherichia coli was cultured with aeration and stirring in a 10L fermenter at a temperature of 30°C and a pH of 7.0. Stirring and aeration were adjusted to control dissolved oxygen at 25%. After the glycerol in the starting medium was exhausted, the induction medium (lactose: 50 g / L, glycerol: 200 g / L) was added to induce the production of the enzyme. The flow rate is 60-250mL / hour, and the maximum flow rate is gradually reached within 3 hours. Temper...

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Abstract

The invention provides a method for catalyzing chenodeoxycholic acids to compound ursodesoxycholic acids through efficient whole-cells. A 7a-hydroxysteroid dehydrogenase (7a-HSDH) and a lactic dehydrogenase (LDH) for regeneration of coenzyme nicotinamide adenine dinucleotide (NAD) are efficiently co-expressed in escherichia coli, escherichia coli whole cells are used to catalyze chenodeoxycholic acids (CDCA) to generate 3 alpha (Alpha)-hydroxyl-7-oxo-5bata (Beta)- cholanic acids (7-KLCA), and a reaction liquid which is obtained by catalyzing the chenodeoxycholic acids through whole cells is adjusted to be 7-KLCA crude products. Reconstitution cells can be easily obtained in low cost through a fermentation process, are better than a chemical synthesis method in production cost and product quality, and are suitable for commercial process.

Description

Technical field: [0001] The invention provides a method for efficiently synthesizing ursodeoxycholic acid from chenodeoxycholic acid by whole cells. The invention belongs to the technical field of bioengineering. Background technique: [0002] Ursodeoxycholic acid (I, UDCA) is the main active ingredient contained in the precious traditional Chinese medicine bear bile, and it and its corresponding diastereoisomer chenodeoxycholic acid (II, CDCA) are clinically used to treat various Gallstone disease, various acute and chronic liver diseases, has good effect. The yield of extracting UDCA from bear bile of artificially raised bears is low, the source is limited, and it is against animal protection, so artificial synthesis of UDCA is of great significance. The synthesis methods of UDCA mainly include the method of combining total chemical synthesis and chemical enzymatic method, and the starting material is animal-derived cholic acid (CA) or deoxycholic acid (such as CDCA). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N1/21C12P33/06C12R1/19
Inventor 刘志斌刘经辉吴庆斌周敦秀
Owner 苏州天绿生物制药有限公司
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